搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

CD8+ T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. Although in vitro-polarized,transferred IL-9-secreting CD8+ Tc9 (cytotoxic T lymphocyte subset 9) cells exert greater persistence and antitumor efficacy than Tc1 cells,the underlying mechanism remains unclear. Here,we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models,which is strongly correlated with their persistence. Using RNA-sequence and functional validation,we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3,upregulated fatty acid oxidation and mitochondrial activity,and rendered Tc9 cells with reduced lipid peroxidation and resistance to tumor- or ROS-induced ferroptosis in the tumor microenvironment. IL-9 signaling deficiency,inhibiting STAT3,or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells,resulting in impaired longevity and antitumor ability. Similarly,human Tc9 cells also exhibited lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8+ T cells expressed lower IL9 and higher lipid peroxidation- and ferroptosis-related genes than circulating CD8+ T cells in patients with melanoma. This study indicates that lipid peroxidation regulates Tc9 cell longevity and antitumor effects via the IL-9/STAT3/fatty acid oxidation pathway and regulating T cell lipid peroxidation can be used to enhance T cell-based immunotherapy in human cancer. View Publication

BACKGROUND Acute respiratory distress syndrome (ARDS) is a neutrophil-associated disease. Delayed neutrophil apoptosis and increased levels of neutrophil extracellular traps (NETs) have been described in ARDS. We aimed to investigate the relationship between these phenomena and their potential as inflammation drivers. We hypothesized that delayed neutrophil apoptosis might enhance NET formation in ARDS. METHOD Our research was carried out in three aspects: clinical research,animal experiments,and in vitro experiments. First,we compared the difference between neutrophil apoptosis and NET levels in healthy controls and patients with ARDS and analyzed the correlation between neutrophil apoptosis and NET levels in ARDS. Then,we conducted animal experiments to verify the effect of neutrophil apoptosis on NET formation in Lipopolysaccharide-induced acute lung injury (LPS-ALI) mice. Furthermore,this study explored the relationship between neutrophil apoptosis and NETs at the cellular level. Apoptosis was assessed using morphological analysis,flow cytometry,and western blotting. NET formation was determined using immunofluorescence,PicoGreen assay,SYTOX Green staining,and western blotting. RESULTS ARDS neutrophils lived longer because of delayed apoptosis,and the cyclin-dependent kinase inhibitor,AT7519,reversed this phenomenon both in ARDS neutrophils and neutrophils in bronchoalveolar lavage fluid (BALF) of LPS-ALI mice. Neutrophils in a medium containing pro-survival factors (LPS or GM-CSF) form more NETs,which can also be reversed by AT7519. Tissue damage can be reduced by promoting neutrophil apoptosis. CONCLUSIONS Neutrophils with extended lifespan in ARDS usually enhance NET formation,which aggravates inflammation. Enhancing neutrophil apoptosis in ARDS can reduce the formation of NETs,inhibit inflammation,and consequently alleviate ARDS. View Publication

New strategies that augment T cell responses are required to broaden the therapeutic arsenal against cancer. CD96,TIGIT,and CD226 are receptors that bind to a communal ligand,CD155,and transduce either inhibitory or activating signals. The function of TIGIT and CD226 is established,whereas the role of CD96 remains ambiguous. Using a panel of engineered antibodies,we discovered that the T cell stimulatory activity of anti-CD96 antibodies requires antibody cross-linking and is potentiated by Fc$\gamma$ receptors. Thus,soluble Fc silent" anti-CD96 antibodies failed to stimulate human T cells whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and it was dependent on antibody trans-cross-linking by Fc$\gamma$RI. In contrast neither human IgG2 nor variants with increased Fc$\gamma$ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T cell activation leading to proliferation cytokine secretion and resistance to Treg suppression. Furthermore CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its cross-linking activated tumor-infiltrating T cells thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy." View Publication

BACKGROUND Chronic kidney disease patients are at increased risk of mortality with cardiovascular diseases and infections as the two leading causes of death for end-stage kidney disease treated with hemodialysis (HD). Mortality from bacterial infections in HD patients is estimated to be 100-1000 times higher than in the healthy population. METHODS We comprehensively characterized highly pure circulating neutrophils from HD and healthy donors. RESULTS Protein levels and transcriptome of HD patients' neutrophils indicated massive neutrophil degranulation with a dramatic reduction in reactive oxygen species (ROS) production during an oxidative burst and defective oxidative cellular signaling. Moreover,HD neutrophils exhibit severely impaired ability to generate extracellular NET formation (NETosis) in NADPH oxidase-dependent or independent pathways,reflecting their loss of capacity to kill extracellular bacteria. Ectopic hydrogen peroxidase (H2O2) or recombinant human SOD-1 (rSOD-1) partly restores and improves the extent of HD dysfunctional neutrophil NET formation. CONCLUSIONS Our report is one of the first singular examples of severe and chronic impairment of NET formation leading to substantial clinical susceptibility to bacteremia that most likely results from the metabolic and environmental milieu typical to HD patients and not by common human genetic deficiencies. In this manner,aberrant gene expression and differential exocytosis of distinct granule populations could reflect the chronic defect in neutrophil functionality and their diminished ability to induce NETosis. Therefore,our findings suggest that targeting NETosis in HD patients may reduce infections,minimize their severity,and decrease the mortality rate from infections in this patient population. View Publication

BACKGROUND Novel therapies are urgently needed for ovarian cancer (OC),the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA,including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling,recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change,which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors,and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response. METHODS Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1,anti-CD8,or anti-NK1.1 antibodies every 3 days. RESULTS We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-$\beta$ compared with either perturbation alone. Furthermore,DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells. CONCLUSION In summary,we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus,epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC,a disease that does not respond to current immunotherapies. View Publication

The blood?brain barrier (BBB) is a critical selective interface between the central nervous system (CNS) and the blood circulation. BBB dysfunction plays an important role in the neurological damage caused by sepsis. However,the mechanisms underlying the disruption of the BBB during sepsis remain unclear. We established a human induced pluripotent stem cell (iPSC)-derived BBB model and reported that treating with sepsis patient serum leads to structural and functional disruption of the BBB. In a cecal ligation and puncture (CLP)-induced mouse model of sepsis,we also observed disruption of the BBB,inflammation in the brain,and impairments in cognition. In both models,we found that the expression of TREM-1 was significantly increased in endothelial cells. TREM-1 knockout specifically in endothelial cells alleviated BBB dysfunction and cognitive impairments. Further study revealed that TREM-1 affects the expression of genes involved in the PI3K/Akt signaling pathway. The protective effects of TREM-1 inhibition on the BBB and cognition were abrogated by PI3K inhibitors. Our findings suggest that endothelial TREM-1 induces sepsis-induced BBB disruption and cognitive impairments via the PI3K/Akt signaling pathway. Targeting endothelial TREM-1 or the PI3K/Akt signaling pathway may be a promising strategy to maintain BBB integrity and improve cognitive function in sepsis patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03469-5. View Publication

The work highlights the different calcium channels involved in controlling protein synthesis in neurons,and shows the dysfunction of this process in Alzheimer’s disease neurons. Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity–mediated protein synthesis,another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases—increased translation inhibition,followed by a decrease in translation inhibition,and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition,which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs,RyRs,and SOCE. Furthermore,we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally,we signify the relevance of our findings in the context of Alzheimer’s disease. Using neurons derived from human fAD iPSCs and transgenic AD mice,we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis,and its implications in neurodegeneration. View Publication

Understanding the multicellular organization of stem cells is vital for determining the mechanisms that coordinate cell fate decision-making during differentiation; these mechanisms range from neighbor-to-neighbor communication to tissue-level biochemical gradients. Current methods for quantifying multicellular patterning tend to capture the spatial properties of cell colonies at a fixed scale and typically rely on human annotation. We present a computational pipeline that utilizes topological data analysis to generate quantitative,multiscale descriptors which capture the shape of data extracted from 2D multichannel microscopy images. By applying our pipeline to certain stem cell colonies,we detected subtle differences in patterning that reflect distinct spatial organization associated with loss of pluripotency. These results yield insight into putative directed cellular organization and morphogen-mediated,neighbor-to-neighbor signaling. Because of its broad applicability to immunofluorescence microscopy images,our pipeline is well-positioned to serve as a general-purpose tool for the quantitative study of multicellular pattern formation. View Publication

BackgroundThree common isoforms of the apolipoprotein E (APOE) gene - APOE2,APOE3,and APOE4 - hold varying significance in Alzheimer’s Disease (AD) risk. The APOE4 allele is the strongest known genetic risk factor for late-onset Alzheimer’s Disease (AD),and its expression has been shown to correlate with increased central nervous system (CNS) amyloid deposition and accelerated neurodegeneration. Conversely,APOE2 is associated with reduced AD risk and lower CNS amyloid burden. Recent clinical data have suggested that increased blood-brain barrier (BBB) leakage is commonly observed among AD patients and APOE4 carriers. However,it remains unclear how different APOE isoforms may impact AD-related pathologies at the BBB.MethodsTo explore potential impacts of APOE genotypes on BBB properties and BBB interactions with amyloid beta,we differentiated isogenic human induced pluripotent stem cell (iPSC) lines with different APOE genotypes into both brain microvascular endothelial cell-like cells (BMEC-like cells) and brain pericyte-like cells. We then compared the effect of different APOE isoforms on BBB-related and AD-related phenotypes. Statistical significance was determined via ANOVA with Tukey’s post hoc testing as appropriate.ResultsIsogenic BMEC-like cells with different APOE genotypes had similar trans-endothelial electrical resistance,tight junction integrity and efflux transporter gene expression. However,recombinant APOE4 protein significantly impeded the “brain-to-blood” amyloid beta 1–40 (A?40) transport capabilities of BMEC-like cells,suggesting a role in diminished amyloid clearance. Conversely,APOE2 increased amyloid beta 1–42 (A?42) transport in the model. Furthermore,we demonstrated that APOE-mediated amyloid transport by BMEC-like cells is dependent on LRP1 and p-glycoprotein pathways,mirroring in vivo findings. Pericyte-like cells exhibited similar APOE secretion levels across genotypes,yet APOE4 pericyte-like cells showed heightened extracellular amyloid deposition,while APOE2 pericyte-like cells displayed the least amyloid deposition,an observation in line with vascular pathologies in AD patients.ConclusionsWhile APOE genotype did not directly impact general BMEC or pericyte properties,APOE4 exacerbated amyloid clearance and deposition at the model BBB. Conversely,APOE2 demonstrated a potentially protective role by increasing amyloid transport and decreasing deposition. Our findings highlight that iPSC-derived BBB models can potentially capture amyloid pathologies at the BBB,motivating further development of such in vitro models in AD modeling and drug development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-024-00580-2. View Publication

IntroductionGlucose Transporter 1-Deficiency Syndrome (GLUT1-DS) is a rare genetic disorder caused by mutations in the gene encoding for GLUT1 and characterized by impaired glucose uptake in the brain. This leads to brain hypometabolism and the development of symptoms that include epilepsy,motor dysfunctions and cognitive impairment. The development of patient-specific in vitro models is a valuable tool for understanding the pathophysiology of rare genetic disorders and testing new therapeutic interventions.MethodsIn this study,we generated brain organoids from induced pluripotent stem cells (iPSCs) derived either from a GLUT1-DS patient or a healthy individual. The functional organoids were analyzed for cellular composition,maturity,and electrophysiological activity using a custom-made microelectrode array (MEA) platform,which allowed for the detection of spikes,burst patterns,and epileptiform discharges.ResultsImmunostaining revealed a similar distribution of neurons and astrocytes in both healthy and GLUT1-DS brain organoids,though GLUT1-DS brain organoids exhibited reduced cellular density and smaller overall size. Electrophysiological recordings demonstrated functional spike profiles in both organoid types. Notably,our study demonstrates that brain organoids derived from a GLUT1-DS patient exhibit distinct epileptiform activity and heightened sensitivity to glucose deprivation,reflecting key features of the disorder.DiscussionThese findings validate the use of brain organoids as a model for studying GLUT1-DS and highlight their potential for testing novel therapeutic strategies aimed at improving glucose metabolism and managing epilepsy in patients. View Publication